The present invention is directed to a pharmaceutical composition containing refined detoxified endotoxin (RDE), cell wall skeleton (CWS) and trehalose dimycolate (TDM). The RDE used in the present composition is characterized as having no detectable 2-keton-3-deoxyoctanoate, between about 350 and 475 nmoles/mg of phosphorus and between about 1700 and 2000 nmoles/mg of fatty acids. The composition is effective to obtain remission and/or regression of cancerous tumors in warm-blooded animals.
Endotoxic extracts obtained from Enterobacteriaciae including parent organisms and mutants are known. These extracts have been used for immunotherapy of various immunogenic tumors [see, Peptides as Requirement for Immunotherapy of the Guinea-Pig Line-10 Tumor with Endotoxins; Ribi, et al, Cancer Immunol. Immunother. Vol. 7, pgs. 43-58 (1979) incorporated herein by reference]. However, the endotoxin extracts are known to be highly toxic and, therefore, of limited use in the treatment of cancerous tumors. Efforts have been made to "detoxify" the endotoxins while retaining its tumor regressive capacity. As shown, in Ribi, et al, chemical procedures known to detoxify endotoxins while retaining adjuvanticity, such as succinylation and phthalylation resulted in both loss of endotoxicity and tumor regressive potency. Therefore, prior art attempts to obtain an endotoxin product having high tumor regressive potency and little or no toxicity have thus far not been successful.
The combination of cell wall skeleton and trehalose dimycolate is known in the art (see, Biologically Active Components from Mycobacterial Cell Walls. II. Supression and Regression of Strain-2 Guinea Pig Hepatoma; Meyer, et al, Journal of the National Cancer Institute, Vol. 52, No. 1, January, 1974; and Mycobacterial Cell Wall Components in Tumor Supression and Regression; Ribi, et al, National Cancer Institute Monograph No. 39, pgs. 115-120, October, 1972.
Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan mucopeptide containing remnants of trehalose mycolate ("P.sub.3 ") and undigested tuberculoproteins. Cell wall skeleton is obtained from any mycobacteria including, but not limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M.kansasii, M.tuberculosis (Strain H37 RV and Ayoma B), and M.bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such nonmycobacteria as E.coli, B.abortus and Coxiella burnettii.
Cell wall skeleton is produced by first growing and harvesting bacteria such as M.bovis, Strain BCG (bacillus Calmette-Guerin). The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells separating the outer envelope or cell wall from the protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g. trypsin and/or chymotrypsin) to give purified cell wall skeleton.
Trehalose dimycolate (TDM) may be obtained from the following organisms as, for example, M.avium, M.phlei, M.tuberculosis (Strain H37 RV and Ayoma B), M.bovis BCG, M.smegmatis, M.kansasii, Nocardia rubra and Corynebacterium diphtheriae.
Bacteria, such as M.avium, are grown, harvested and then heat killed. The cell mass is extracted with several solvents and then an active, solvent soluble fraction is extracted. This extract is further purified by a series of solvent extractions to provide crude TDM (see, Biologically Active Components from Mycobacterial Cell Walls. I. Isolation and Composition of Cell Wall Skeleton and Component P.sub.3 ; Azuma, et al, Journal of the National Cancer Institute, Vol. 52, pps. 95-101, 1974 incorporated herein by reference). As disclosed in Azuma et al, crude TDM may then be further purified by centrifugal microparticulate silica gel chromatography to give purified TDM.
CWS and TDM produced and described above can be used as an oil droplet emulsion to obtain an antitumor composition suitable for injection (see, Immunotherapy With Non-Viable Microbial Components; Ribi et al; Annals of the New York Academy Of Science, Vol. 227, pps. 228-238, Sept. 20, 1976).
It is therefore an object of the invention to provide a pharmaceutical composition comprising a refined detoxified endotoxin, cell wall skeleton and trehalose dimycolate which is effective in the treatment of cancerous tissues in warm blooded animals and humans.
It is another object of the invention to provide a pharmaceutical composition containing these components without the deleterious side effects normally associated with endotoxins.